U.S. patent application number 17/365733 was filed with the patent office on 2021-10-28 for design and development of neurokinin-1 receptor-binding agent delivery conjugates.
The applicant listed for this patent is Purdue Research Foundation. Invention is credited to Ananda Kumar Kanduluru, Philip Stewart Low.
Application Number | 20210330819 17/365733 |
Document ID | / |
Family ID | 1000005695986 |
Filed Date | 2021-10-28 |
United States Patent
Application |
20210330819 |
Kind Code |
A1 |
Kanduluru; Ananda Kumar ; et
al. |
October 28, 2021 |
DESIGN AND DEVELOPMENT OF NEUROKININ-1 RECEPTOR-BINDING AGENT
DELIVERY CONJUGATES
Abstract
Neurokinin-1 (NK-1) receptor-binding agent delivery conjugates,
compositions comprising NK-1 receptor-binding agent delivery
conjugates, and methods for making and administering NK-1
receptor-binding agent delivery conjugates are provided. A
conjugate may include an NK-1 receptor-binding moiety, a tinker
group containing at least one linker selected from the group of a
releasable linker and a spacer linker, and an active agent linked
to the linker group. The active agent may be selected from the
group of fluorophore-containing compounds, radionuclide-containing
compounds, and therapeutic agents for treatment of tumor cells
characterized by over-expression of the NK-1 receptor.
Inventors: |
Kanduluru; Ananda Kumar;
(West Lafayette, IN) ; Low; Philip Stewart; (West
Lafayette, IN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Purdue Research Foundation |
West Lafayette |
IN |
US |
|
|
Family ID: |
1000005695986 |
Appl. No.: |
17/365733 |
Filed: |
July 1, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15501630 |
Feb 3, 2017 |
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PCT/US15/44229 |
Aug 7, 2015 |
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17365733 |
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62035427 |
Aug 9, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 49/0041 20130101;
A61K 51/0455 20130101; A61K 51/0478 20130101; A61K 51/0482
20130101; C09B 23/0025 20130101; A61K 38/05 20130101; C09B 11/24
20130101; A61K 51/06 20130101; A61K 31/475 20130101; A61K 51/0459
20130101; A61K 51/08 20130101; A61K 49/0052 20130101; C07K 14/70571
20130101; A61K 31/404 20130101; C09B 23/0066 20130101; A61K 31/4545
20130101 |
International
Class: |
A61K 49/00 20060101
A61K049/00; C07K 14/705 20060101 C07K014/705; C09B 23/01 20060101
C09B023/01; C09B 11/24 20060101 C09B011/24; A61K 31/404 20060101
A61K031/404; A61K 51/04 20060101 A61K051/04; A61K 31/4545 20060101
A61K031/4545; A61K 31/475 20060101 A61K031/475; A61K 38/05 20060101
A61K038/05; A61K 51/06 20060101 A61K051/06; A61K 51/08 20060101
A61K051/08 |
Claims
1-15. (canceled)
16. A neurokinin-1 (NK-1) receptor-binding conjugate comprising: an
NK-1 receptor-binding moiety; an active agent comprising a
radionuclide; and a linker, which links the NK-1 receptor-binding
moiety and the active agent, comprises at least one spacer linker,
and optionally further comprises a releasable linker.
17. The NK-1 receptor-binding conjugate of claim 16, wherein the
active agent is a radio-imaging agent, an optical imaging agent, a
position emission tomography (PET)imaging agent, a magnetic
resonance imaging (MRI) contrast agent, a computed tomography (CT)
contrast agent, or a fluorescence resonance energy transfer (FRET)
imaging agent.
18. The NK-1 receptor-binding conjugate of claim 16, wherein the
active agent comprises a chelator in complex with a radionuclide
selected from the group consisting of technetium-99m (.sup.99mTc),
gallium-68 (.sup.68Ga), indium-111 (.sup.111In), yttrium-90
(.sup.90Y), lutetium-177 (.sup.177Lu), zirconium-89 (.sup.89Zr),
actinium-225 (.sup.225Ac), cobalt-60 (.sup.60Co), and copper-64
(.sup.64Cu).
19. The NK-1 receptor-binding conjugate of claim 16, wherein the
radionuclide is .sup.99mTc or .sup.64Cu.
20. The NK-1 receptor-binding conjugate of claim 18, wherein the
chelator is selected from the group consisting of: ##STR00030##
wherein each R is independently H, alkyl, heteroalkyl, cycloalkyl,
heterocyclyl, alkenyl, alkynyl, aryl, heteroaryl, arylalkyl, or
heteroarylalkyl, each of which is optionally substituted, and
wherein one R comprises a heteroatom, which is attached to the
linker; X is oxygen, nitrogen, or sulfur, and is attached to the
linker; and n is an integer from 1 to 5.
21. The NK-1 receptor-binding conjugate of claim 20, wherein the
heteroatom in R is oxygen, nitrogen or sulfur.
22. The NK-1 receptor-binding conjugate of claim 19, wherein the
chelator is a tripeptide or tetrapeptide.
23. The NK-1 receptor-binding conjugate of claim 22, wherein the
tripeptide has the formula: ##STR00031## wherein each R is
independently H, alkyl, heteroalkyl, cycloalkyl, heterocyclyl,
alkenyl, alkynyl, aryl, heteroaryl, arylalkyl, or heteroarylalkyl,
each of which is optionally substituted, and wherein one R
comprises a heteroatom, which is attached to the linker.
24. The NK-1 receptor-binding conjugate of claim 23, wherein the
heteroatom in R is oxygen, nitrogen or sulfur.
25. The NK-1 receptor-binding conjugate of claim 18, wherein the
chelator is selected from the group consisting of: ##STR00032##
26. The NK-1 receptor-binding conjugate of claim 16, wherein the
NK-1 receptor-binding moiety comprises a selective NK-1 receptor
antagonist or a derivative thereof.
27. The NK-1 receptor-binding conjugate of claim 16, wherein the
NK-1 receptor-binding moiety is selected from the group consisting
of: ##STR00033##
28. The NK-1 receptor-binding conjugate of claim 16, wherein the
spacer linker comprises amino acids selected from the group
consisting of naturally occurring amino acids and stereoisomers
thereof.
29. The NK-1 receptor-binding conjugate of claim 16, wherein the
linker comprises dithioalkyloxycarbonyl.
30. The NK-1 receptor-binding conjugate of claim 16, wherein the
linker comprises 3-thiosuccinimid-1-ylalkyloxy.
31. The NK-1 receptor-binding conjugate of claim 16, wherein the
linker comprises 3-cysteinylsuccinimid-1-ylalkyloxy, wherein the
cysteinyl is optically active or optically inactive.
32. The NK-1 receptor-binding conjugate of claim 16, wherein the
conjugate is selected from: ##STR00034##
33. A pharmaceutical composition comprising a NK-1 receptor-binding
conjugate of claim 16, and a pharmaceutically acceptable carrier,
diluent, or excipient.
34. A method of using a NK-1 receptor-binding conjugate, which
method comprises: (a) administering to a subject an effective
amount of the NK-1 receptor-binding conjugate of claim 16,
optionally as a pharmaceutical composition comprising the NK-1
receptor-binding conjugate and a pharmaceutically acceptable
carrier, diluent, or excipient, and (b) detecting the radionuclide
in the subject.
35. The method of claim 34, which further comprises diagnosing a
disease, locating metastatic disease, detecting disease recurrence,
or monitoring response to therapy.
36. The method of claim 35, which further comprises selecting a
patient for cholecystokinin 2 receptor targeted therapy.
Description
BACKGROUND
[0001] The mammalian immune system provides mechanisms for the
recognition and elimination of tumor cells invading foreign
pathogens. While the immune system normally provides a strong line
of defense, tumor cells or pathogens may nonetheless evade a host
immune response and proliferate or persist with concomitant host
pathogenicity. Chemotherapeutic agents and radiation therapies have
been developed to eliminate, for example, replicating neoplasms.
However, many of the currently available chemotherapeutic agents
and radiation therapy regimens have adverse side effects by
likewise affecting normal host cells, such as cells of the
hematopoietic system. The adverse side effects of these anticancer
drugs highlight the need for the development of new therapies
selective for pathogenic cell populations and with reduced host
toxicity. Moreover, the target selectiveness can be implemented to
improve other therapeutic and diagnostic techniques that are
tailored to a pathogenic cell population.
[0002] Researchers have developed therapeutic protocols for
destroying pathogenic cells by targeting cytotoxic compounds to
such cells. Many of these protocols utilize toxins or
chemotherapeutic agents conjugated to antibodies that bind to
antigens that are unique to, or over-expressed by, the pathogenic
cells in an attempt to minimize delivery of the toxin to normal
cells.
[0003] The neurokinin-1 (NK-1) receptor (also known as the
tachykinin receptor 1 or SP receptor) is a G protein-coupled
receptor that is the product of the TACR1 gene. The NK-1 receptor
is 407 amino acid residues, and, along with other tachykinin
receptors, is made of seven hydrophobic transmembrane domains with
three extracellular and three intracellular loops, an
amino-terminus, and a cytoplasmic carboxy-terminus. The loops have
functional sites, including two cysteines amino acids for a
disulfide bridge, Asp-Arg-Tyr that is responsible for association
with arrestin, and Lys/Arg-Lys/Arg-X-X-Lys/Arg, which interacts
with G-proteins. While the NK-1 receptor has some affinity for all
tachykinins, its endogenous ligand is the peptide Substance P
(SP).
[0004] Upon binding to the NK-1 receptor, SP has been shown to
induce changes such as tumor cell proliferation, angiogenesis, and
migration of the tumor cells for metastasis. In contrast, NK-1
receptor antagonists exert antiproliferative effects on
NK-1-receptor expressing cells by inducing apoptosis. Moreover, it
is known that NK-1 receptors are overexpressed in some tumors, and
that tumor cells express several isoforms of the NK-1 receptor. For
example, NK-1 receptor over-expression has been reported in certain
cancers of the larynx, stomach, colon, pancreas, and breast as well
as glioblastomas, gliomas, astrocytomas, melanomas,
retinoblastomas, and neuroblastomas.
[0005] All of these data suggest that NK-1 receptor expression may
play an important role in the development of cancer, that SP may be
a universal mitogen in NK-1 receptor-expressing tumor cells, and/or
that expression of the NK-1 receptor may be utilized to diagnose or
identify specific tumors. Further, the data suggest that NK-1
receptor antagonists could offer a promising therapeutic strategy
for the treatment of human cancer, since they act as broad-spectrum
antitumor agents. In sum, the NK-1 receptor may be a new and
promising target in the diagnosis and/or treatment of human
cancer.
SUMMARY
[0006] The various embodiments provide compositions and methods for
making and administering a neurokinin-1 (NK-1) receptor-binding
agent delivery conjugate that includes an NK-1 receptor-binding
moiety, a linker group that includes one or more linker selected
form the group of a releasable linker and a spacer linker, and an
active agent linked to the linker group.
[0007] In some embodiments, the linker group includes at least one
spacer linker, and the active agent is selected from the group
consisting of a fluorophore-containing compound and a
radionuclide-containing compound. In some embodiments, the linker
group includes at least one releasable linker, and the active agent
is a therapeutic agent. In some embodiments, the active agent is a
radionuclide-containing compound including one of technetium-99m
and copper-64, and a chelating portion that forms a complex with
the radionuclide.
[0008] In some embodiments, the active agent is a therapeutic agent
for treatment of tumor cells characterized by over-expression of
the NK-1 receptor, in which the therapeutic agent is selected from
the group of tubulysin B hydrazide (TubH) and disulfide-activated
desacetyl vinblastine hydrazide (DAVBH).
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] The accompanying drawings, which are incorporated herein and
constitute part of this specification, illustrate example aspects
of the invention, and together with the general description given
above and the detailed description given below, serve to explain
the features of the invention.
[0010] FIG. 1A is a schematic reaction equation illustrating the
formation of an NK-1 receptor-binding moiety.
[0011] FIG. 1B provides in vivo mice therapeutic data for HEK
293-NK1R tumor xenograft models showing behavior of tumor volumes
from the NK1R-EC20 peptide linker-TubH conjugate.
[0012] FIG. 1C provides in vivo mice therapeutic data on HEK
293-NK1R tumor xenograft models showing behavior body weights
during the therapy shown in FIG. 1B.
[0013] FIG. 2 is a schematic reaction equation illustrating the
synthesis of an NKIRL-Lys peptide-rhodamine conjugate.
[0014] FIG. 3 is a schematic reaction equation illustrating the
synthesis of an NK1RL-EC20 peptide-LS288-maleimide conjugate.
[0015] FIG. 4 is a set of confocal microscopy images showing in
vitro binding of NKIRL-Lys peptide linker-rhodamine to HEK 293-NK1R
cells.
[0016] FIG. 5 is a graph showing the binding of NKIRL-Lys peptide
rhodamine to HEK 293-NK1R cells by flow cytometry.
[0017] FIGS. 6A and 6B are graphs showing binding affinity of two
fluorescent imaging conjugates in cultured HEK 293-NK1R cells
expressing NK-1 receptor.
[0018] FIG. 6C is a set of images showing (a-b) HEK 293-NK1R tumor
xenograft model mice treated with conjugates in which LS288 is the
active agent, (c-d) blocking images for the HEK 293-NK1R tumor
xenograft model mice with the treatment in a-b, and (e-f)
NK1R-negative tumor xenograft model mice treated with conjugates in
which LS288 is the active agent.
[0019] FIG. 6D is a biodistribution study of the mice imaged in
FIG. 6C.
[0020] FIG. 7A is a plot showing the radioactivity binding affinity
of an NK1RL-EC20 peptide linker-.sup.99mTc conjugate versus
concentration of the conjugate in cells.
[0021] FIG. 7B is a set of whole body mice images showing HEK
293-NK1R tumor xenograft model treated with the NK1RL-EC20 peptide
linker-.sup.99.sup.mTc conjugate (left), and showing blocking
(right).
[0022] FIG. 7C is a biodistribution study of the imaged mice from
FIG. 7B for the NK1RL-EC20 peptide linker-.sup.99mTc conjugate and
a competitive NK-1 receptor ligand.
[0023] FIG. 7D is a set of whole body mice images on SPECT-CT for
NK1RL-EC20 peptide linker-.sup.99.sup.mTc conjugate in HEK 293-NK1R
tumor xenograft model mice.
[0024] FIG. 7E is a set of whole body mice images on SPECT-CT for
NK1RL-EC20 peptide linker-.sup.99mTc conjugate in NK1R-negative
tumor xenograft model mice.
[0025] FIG. 8A is a plot showing region of interest (ROI)
radioactivity of NK1RL-PEG2-NOTA-{circumflex over ( )}Cu conjugate
in NK1R-transduced and non-transduced xenografts.
[0026] FIG. 8B is a plot showing the NK1RL-PEG2-NOTA-.sup.64Cu
conjugate uptake ratio between the NK1R-transduced and
non-transduced xenografts in various areas at 20 hours
post-injection.
[0027] FIG. 8C is a set of PET images showing HEK 293-NK1R tumor
xenograft model mice treated with a conjugate in which the active
agent has .sup.64Cu.
[0028] FIG. 9A is a plot showing the radioactivity binding affinity
of an NK1RL-PEG36-based short EC20 linker-".sup.mTc conjugate
versus concentration of the conjugate in cells in the presence and
absence of a competing ligand.
[0029] FIG. 9B is a set of whole body mice images showing HEK
293-NK1R tumor xenograft model treated with the NK1RL-PEG36-based
short EC20 linker-.sup.99.sup.mTc conjugate with shielding (top
row), and without shielding (bottom row).
[0030] FIG. 9C is a biodistribution study of the imaged mice from
FIG. 9B for the NK1RL-PEG36-based short EC20 linker-.sup.99mTc
conjugate and a competitive NK-1 receptor ligand at 2 hours
post-injection.
[0031] FIG. 9D is a biodistribution study of the imaged mice from
FIG. 9B for the NK1RL-PEG36-based short EC20 linker-.sup.99mTc
conjugate and a competitive NK-1 receptor ligand at 8 hours
post-injection.
[0032] FIG. 10A is a schematic reaction equation illustrating the
synthesis of an NK1RL-SF5-Lys peptide linker-rhodamine
conjugate.
[0033] FIG. 10B is a plot showing the fluorescence binding affinity
of an NK1RL-SF5-Lys peptide linker-rhodamine conjugate in cultured
HEK 293-NK1R cells expressing NK-1 receptor.
[0034] FIG. 10C is a set of confocal microscopy images showing in
vitro binding of NK1RL-SF5-Lys peptide linker-rhodamine to HEK
293-NK1R cells.
[0035] FIG. 11 is a schematic reaction equation illustrating the
synthesis of an NK1RL-SF5-tyrosine peptide linker-S0456
conjugate.
[0036] FIG. 12 is a plot showing the fluorescence binding affinity
of an NK1RL-SF5-tyrosine peptide linker-S0456 conjugate versus
concentration of the conjugate in cells in the presence of a
competing ligand.
[0037] FIG. 13A is a PET image showing a HEK 293-NK1R tumor
xenograft model mouse treated with an
NK1RL-PEG2-DOTA-.sup.1.sup.11I.eta. conjugate.
[0038] FIG. 13B is a plot showing the
NK1RL-PEG2-DOTA-.sup.1.sup.1.sup.1in conjugate uptake ratio in the
HEK 293-NK1R tumor xenograft model in various areas at 4 hours
post-injection.
[0039] FIG. 13C is a whole body mouse image on SPECT-CT for
NK1RL-PEG2-DOTA-.sup.1.sup.1.sup.1in conjugate in the HEK 293-NK1R
tumor xenograft mouse.
DETAILED DESCRIPTION
[0040] The present invention may be understood more readily by
reference to the following detailed description taken in connection
with the accompanying figures and examples, which form a part of
this disclosure. It is to be understood that the various
embodiments are not limited to the specific devices, methods,
applications, conditions or parameters described and/or shown
herein, and that the terminology used herein is for the purpose of
describing particular embodiments by way of example only and is not
intended to be limiting.
[0041] It is to be appreciated that certain features that are, for
clarity, described herein in the context of separate embodiments,
may also be provided in combination in a single embodiment.
Conversely, various features that are, for brevity, described in
the context of a single embodiment, may also be provided separately
or in any sub-combination. Further, reference to values stated in
ranges include each and every value within that range.
[0042] As used in this specification and the appended claims, the
singular forms "a," "an," and "the" include plural referents unless
the content clearly dictates otherwise.
[0043] The term "alkyl" as used herein refers to a monovalent
linear chain of carbon atoms that may be optionally branched, such
as methyl, ethyl, propyl, 3-methylpentyl, and the like.
[0044] The term "cycloalkyl" as used herein refers to a monovalent
chain of carbon atoms, a portion of which forms a ring, such as
cyclopropyl, cyclohexyl, 3-ethylcyclopentyl, and the like.
[0045] The term "alkylene" as used herein refers to a bivalent
linear chain of carbon atoms that may be optionally branched, such
as methylene, ethylene, propylene, 3-methylpentylene, and the
like.
[0046] The term "cycloalkylene" as used herein refers to a bivalent
chain of carbon atoms, a portion of which forms a ring, such as
cycloprop-1,1-diyl, cycloprop-1,2-diyl, cyclohex-1,4-diyl,
3-ethylcyclopent-1,2-diyl, 1-methylenecyclohex-4-yl, and the
like.
[0047] The term "heterocycle" as used herein refers to a monovalent
chain of carbon and heteroatoms, wherein the heteroatoms are
selected from nitrogen, oxygen, and sulfur, a portion of which,
including at least one heteroatom, form a ring, such as aziridine,
pyrrolidine, oxazolidine, 3-methoxypyrrolidine, 3-methylpiperazine,
and the like.
[0048] The term "alkoxy" as used herein refers to alkyl as defined
herein combined with a terminal oxygen, such as methoxy, ethoxy,
propoxy, 3-methylpentoxy, and the like.
[0049] The term "halo" or "halogen" refers to fluoro, chloro,
bromo, and iodo.
[0050] The term "aryl" as used herein refers to an aromatic mono or
polycyclic ring of carbon atoms, such as phenyl, naphthyl, and the
like.
[0051] The term "heteroaryl" as used herein refers to an aromatic
mono or polycyclic ring of carbon atoms and at least one heteroatom
selected from nitrogen, oxygen, and sulfur, such as pyridinyl,
pyrimidinyl, indolyl, benzoxazolyl, and the like.
[0052] The term "substituted aryl" or "substituted heteroaryl" as
used herein refers to aryl or heteroaryl substituted with one or
more substituents selected, such as halo, hydroxy, amino, alkyl or
dialkylamino, alkoxy, alkylsulfonyl, cyano, nitro, and the
like.
[0053] The term "iminoalkylidenyl" as used herein refers to a
divalent radical containing alkylene as defined herein and a
nitrogen atom, where the terminal carbon of the alkylene is
double-bonded to the nitrogen atom, such as the formulae
--(CH).dbd.N--, --(CH.sub.2).sub.2(CH).dbd.N--,
--CH.sub.2C(Me)=N--, and the like.
[0054] The term "amino acid" as used herein refers generally to
aminoalkylcarboxylate, where the alkyl radical is optionally
substituted with alkyl, hydroxy alkyl, sulfhydrylalkyl, aminoalkyl,
carboxyalkyl, and the like, including groups corresponding to the
naturally occurring amino acids, such as serine, cysteine,
methionine, aspartic acid, glutamic acid, and the like.
[0055] The term "arylalkyl" refers to aryl as defined herein
substituted with an alkylene group, as defined herein, such as
benzyl, phenethyl, a-methylbenzyl, and the like.
[0056] It should be understood that the above-described terms can
be combined to generate chemically-relevant groups, such as
"alkoxyalkyl" referring to methyloxymethyl, ethyloxyethyl, and the
like, and "haloalkoxyalkyl" referring to trifluoromethyloxyethyl,
1,2-difluoro-2-chloroeth-1-yloxypropyl, and the like.
[0057] The term "amino acid derivative" as used herein refers
generally to aminoalkylcarboxylate, where the amino radical or the
carboxylate radical are each optionally substituted with alkyl,
carboxylalkyl, alkylamino, and the like, or optionally protected;
and the intervening divalent alkyl fragment is optionally
substituted with alkyl, hydroxy alkyl, sulfhydrylalkyl, aminoalkyl,
carboxyalkyl, and the like, including groups corresponding to the
side chains found in naturally occurring amino acids, such as are
found in serine, cysteine, methionine, aspartic acid, glutamic
acid, and the like.
[0058] The term "peptide" as used herein refers generally to a
series of amino acids and amino acid analogs and derivatives
covalently linked one to the other by amide bonds.
[0059] The term "releasable linker" as used herein refers to a
linker that includes at least one bond that can be broken under
physiological conditions (e.g., a pH-labile, acid-labile,
redox-labile, or enzyme-labile bond). It should be appreciated that
such physiological conditions resulting in bond breaking include
standard chemical hydrolysis reactions that occur, for example, at
physiological pH, or as a result of compartmentalization into a
cellular organelle such as an endosome having a lower pH than
cytosolic pH.
[0060] The term "spacer linker" as used herein refers to an organic
moiety that separates the active agent or releasable linker from
the NK-1 receptor-binding moiety.
[0061] In cancer treatment, the use of conventional
chemotherapeutics may be limited due to their indiscriminate
accumulation in both cancer and healthy cells, thereby resulting in
dose-limiting toxicities in untargeted healthy tissues. One
mechanism to overcome such general accumulation may be selective
ligand-targeted delivery of cytotoxic agents to malignant tissues.
In particular, various embodiments provide for uses of NK-1
receptor-targeted ligands to provide therapeutic and imaging agents
for use in diagnosis and treatment of cancer.
[0062] The various embodiments provide NK-1 receptor-binding agent
delivery conjugates that enable improved mechanisms for targeted
delivery of active agents to cells expressing NK-1 receptors. In
particular, the various embodiment compounds may include an NK-1
receptor-binding moiety (NK), at least one linker group (L), and at
least one active agent (A) (e.g., a drug, a fluorescent dye, a
radioimaging agent, and/or a combination thereof). In the various
embodiments, the NK-1 receptor-binding moiety and the active agent
may be bound to the linker group.
[0063] The linker groups in the various embodiments may include one
or more spacer linkers and releasable linkers, and combinations
thereof, in any order. The various active agents and linker groups
discussed below are provided as non-limiting examples, for which
alternatives are described in International Published Patent
Application No. WO2013/126797, the disclosure which is incorporated
herein by reference in its entirety.
Active Agents
[0064] The active agents D of the conjugates of the present
invention may, in various embodiments, be therapeutic agents and/or
imaging agents. The only limitation on suitable therapeutic agents
and imaging agents is the requirement that they have a position on
the molecule to which can be conjugated the linker L. or that they
can be derivatized to possess such a position without losing the
activity of the active moiety or compromising the ability of the
NK-1 receptor-binding moiety to bind to its receptor with high
affinity.
[0065] The therapeutic agents described herein function through any
of a large number of mechanisms of action. Generally, therapeutic
agents disrupt cellular mechanisms that are important for cell
survival and/or cell proliferation and/or cause apoptosis. By way
of example only, the therapeutic agents can be any compound known
in the art which is cytotoxic, enhances tumor permeability,
inhibits tumor cell proliferation, promotes apoptosis, decreases
anti-apoptotic activity in target cells, is used to treat diseases
caused by infectious agents, enhances an endogenous immune response
directed to the pathogenic cells, or is useful for treating a
disease state caused by any type of pathogenic cell.
[0066] Therapeutic agents suitable for use in accordance with this
invention include, without limitation, adrenocorticoids and
corticosteroids, alkylating agents, antiandrogens, antiestrogens,
androgens, aclamycin and aclamycin derivatives, estrogens,
antimetabolites such as cytosine arabinoside, purine analogs,
pyrimidine analogs, and methotrexate, busulfan, carboplatin,
chlorambucil, cisplatin and other platinum compounds, taxanes, such
as tamoxiphen, taxol, paclitaxel, paclitaxel derivatives,
Taxotere.RTM., and the like, maytansines and analogs and
derivatives thereof, cyclophosphamide, daunomycin, doxorubicin,
rhizoxin, T2 toxin, plant alkaloids, prednisone, hydroxyurea,
teniposide, mitomycins, discodermolides, microtubule inhibitors,
epothilones, tubulysin (e.g., tubulysin B hydrazide), cyclopropyl
benz[e]indolone, seca-cyclopropyl benz[e]indolone,
O--Ac-seca-cyclopropyl benz[e]indolone, bleomycin and any other
antibiotic, nitrogen mustards, nitrosureas, vincristine,
vinblastine, and analogs and derivative thereof such as desacetyl
vinblastine monohydrazide, colchicine, colchicine derivatives,
allocolchicine, thiocolchicine, trityl cysteine, Halicondrin B,
dolastatins such as dolastatin 10, amanitins such as a-amanitin,
camptothecin, irinotecan, and other camptothecin derivatives
thereof, geldanamycin and geldanamycin derivatives, estramustine,
nocodazole, MAP4, colcemid, inflammatory and proinflammatory
agents, peptide and peptidomimetic signal transduction inhibitors,
and any other art-recognized drug or toxin. Other drugs that can be
used in accordance with the invention include penicillins,
dinitrophenol, fluorescein, CpG oligonucleotides, staurosporine and
the kinase inhibitors, Sutent, resiquimod and other Toll-like
receptor agonists, cephalosporins, vancomycin, erythromycin,
clindamycin, rifampin, chloramphenicol, aminoglycoside antibiotics,
gentamicin, amphotericin B, acyclovir, trifluridine, ganciclovir,
zidovudine, amantadine, ribavirin, and any other art-recognized
antimicrobial compound.
[0067] Specific sub-groups of therapeutic agents include, but are
not limited to, radio-therapeutic agents, immunotherapeutic agents,
photodynamic therapy agents and chemo therapeutic agents. The
skilled artisan will understand that there is a wide variety of
radio-therapeutics that will be suitable for use in the conjugates
of the present invention. Suitable examples include, but are not
limited to, .sup.90Y, .sup.31I, .sup.1.sup.7.sup.7Lu,
.sup.6.sup.7Cu, .sup.U.sup.1ln, .sup.186Re, .sup.21'At, and
{circumflex over ( )}Ra.
[0068] The skilled artisan will also understand that there is a
wide variety of chemotherapeutics that will be suitable for use in
the conjugates of the present invention. Suitable examples include,
but are not limited to, tubulysin B hydrazide and desacetyl
vinblastine monohydrazide, calicheamycin, auristatin,
maytansinoids, and any other cytotoxic agent with IC50 value below
10 nM.
[0069] It should also be appreciated that the ligand can be used to
target a nanomedicines or nanoparticle, including but not limited
to a liposome, a lipoplex, a polyplex, a dendrimer, a polymer, a
nanoparticle, or a virus. It should further be recognized that the
aforementioned particles might serve as carriers for DNA, RNA,
siRNA, peptides, proteins, and other biologies.
[0070] Imaging agents suitable for use in the conjugates of the
invention include, but are not limited to, radio-imaging agents,
optical imaging agents, PET imaging agents, MRI contrast agents, CT
contrast agents, and FRET imaging agents, and other agents that may
be used to detect or visualize a tumor, cancer or transformed cell,
whether in vitro, in vivo and ex vivo.
[0071] Applications for conjugates comprising radio-imaging agents
include, but are not limited to, diagnosis of disease and or
locating metastatic disease, detecting disease recurrence following
surgery, monitoring response to therapy, development of a
radio-therapeutic conjugate and selecting patients for subsequent
CCK2R targeted therapy. Radio-imaging agents include radioactive
isotopes, such as a radioactive isotope of a metal, coordinated to
a chelating group. Illustrative radioactive metal isotopes include
technetium, rhenium, gallium, gadolinium, indium, copper, and the
like, including isotopes .sup.U.sup.1ln, .sup.99mTc,
.sup.6.sup.4Cu, .sup.6.sup.7Cu, .sup.67Ga, .sup.6.sup.8Ga, and the
like, or they may include radionuclides that are effective in
radiotherapy.
[0072] Illustratively, the following chelating groups are described
that can be used with the radio-imaging agents:
##STR00001##
wherein R is independently selected in each instance from, for
example, H, alkyl, heteroalkyl, cycloalkyl, heterocyclyl, alkenyl,
alkynyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, and the
like, each of which is optionally substituted, wherein one R
includes a heteroatom, such as nitro, oxygen, or sulfur, and is the
point of attachment of linker L; X is oxygen, nitrogen, or sulfur,
and X is attached to linker L; and n is an integer from 1 to about
5.
[0073] Additional illustrative chelating groups are tripeptide or
tetrapeptides, including but not limited to tripeptides having the
formula:
##STR00002##
wherein R is independently selected in each instance from H, alkyl,
heteroalkyl, cycloalkyl, heterocyclyl, alkenyl, alkynyl, aryl,
heteroaryl, arylalkyl, heteroaryalkyl, and the like, each of which
is optionally substituted. It is to be understood that one R
includes a heteroatom, such as nitro, oxygen, or sulfur, and is the
point of attachment of linker L.
[0074] Applications for conjugates comprising optical imaging
agents include locating and resecting large tumor masses,
delineation of normal and malignant tissue, intraoperative
detection of sentinel lymph nodes, and fluorescent probe for
minimally invasive laparoscopic procedures as an alternative to
second look surgery. The skilled artisan will also understand that
there is a wide variety of optical imaging agents that will be
suitable for use in the conjugates of the present invention. The
only limitations on suitable optical imaging agents is the
requirement that they have a position on the molecule to which can
be conjugated the linker L, or that they can be derivatized to
possess such a position. Examples include, but are not limited to,
Oregon Green fluorescent agents, including but not limited to
Oregon Green 488, Oregon Green 514, and the like, AlexaFluor
fluorescent agents, including but not limited to AlexaFluor 488,
AlexaFluor 647, and the like, fluorescein, and related analogs,
BODIPY fluorescent agents, including but not limited to BODIPY F1,
BODIPY 505, S0456, and the like, rhodamine fluorescent agents,
including but not limited to tetramethylrhodamine, and the like,
near infra-red fluorescent agents, including but not limited to
DyLight 680, DyLight 800, 800CW, LS288, S0456, indocyanine green
and the like, Texas Red, phycoerythrin, and others. Illustrative
optical imaging agents are shown in the following general
structure:
##STR00003##
where X is oxygen, nitrogen, or sulfur, and where X is attached to
linker L; Y is ORa, NRa.sub.2, or NRa.sub.3+; and Y' is 0, NRa, or
NRa.sub.2.sub.+; where each R is independently selected in each
instance from H, fluoro, sulfonic acid, sulfonate, and salts
thereof, and the like; and Ra is hydrogen or alkyl.
[0075] According to another aspect, illustrative optical imaging
agents are shown in the following general structure:
##STR00004##
where X is oxygen, nitrogen, or sulfur, and where X is attached to
linker L; and each R is independently selected in each instance
from H, alkyl, heteroalkyl, and the like; and n is an integer from
0 to about 4.
[0076] The skilled artisan will also understand that there is a
wide variety of PET imaging agents and FRET imaging agents that
will be suitable for use in the conjugates of the present
invention. The only limitations on suitable PET and FRET imaging
agents is the requirement that they have a position on the molecule
to which can be conjugated the linker L, or that they can be
derivatized to possess such a position. Examples of PET imaging
agents include, but are not limited to, .sup.1.sup.8F, .sup.11C,
{circumflex over ( )}Cu, .sup.6.sup.5Cu, and the like. Examples of
FRET imaging agents include, but are not limited to, .sup.64Eu,
.sup.65Eu, and the like. It appreciated that in the case of
.sup.18F and .sup.11C, the imaging isotope may be present on any
part of the linker, or alternatively may be present on a structure
attached to the linker. For example in the case of --F, fluoroaryl
groups, such as fluorophenyl, difluorophenyl, fluoronitrophenyl,
and the like are described. For example in the case of .sup.11C,
alkyl and alkyl aryl are described. Exemplary optical imaging
agents include, but are not limited to, fluorescein (FITC),
rhodamine, LS288, S0456, IR800CW, or another near infrared dye.
Linkers
[0077] Exemplary linkers may include, but are not limited to,
hydrophilic linkers comprised of charged or polar amino acids,
sugars or sugar-containing oligomers, and hydrophilic polymers such
as polyethylene glycol.
[0078] In a first embodiment, the linker L is L1, L2 or L3, where
L1 is HN-Glu-Arg-Asp-CO, L2 is HN-Glu-PS-Glu-PS-CO, and L3 is
HN-Octanoyl-Glu-PS-Glu-PS-CO, in which PS has the following
formula:
##STR00005##
[0079] The linkers used in the production of the embodiment
compounds may also comprise one or more spacer linkers and/or one
or more releasable linkers, and combinations thereof, in any order.
It is appreciated that spacer linkers may be included when
predetermined lengths are selected for separating the NK-1
receptor-binding moiety from the active agent. It is also
appreciated that in certain configurations, releasable linkers may
be included.
[0080] In an example, the linker group may be a releasable linker
that includes a cleavable bond connecting two adjacent atoms.
Following breakage of the cleavable bond, the releasable linker may
be broken into two or more fragments. In another example the
releasable linker may contain a cleavable bond that connects one
end of the releasable linker to other linkers, to the active agent,
or to the NK-1 receptor-binding moiety. Following breakage of the
cleavable bond, the releasable linker may be separated from the
other moiety. Examples of a releasable linker may include, but are
not limited to, a disulfide group and a carbamate group.
[0081] In a particular application, expression of NK-1 receptors
has been reported in a number of lethal cancers of the brain
(glioblastoma, glioma and astrocytoma), skin (melanoma), pancreas,
retina (retinoblastoma), nerve (neuroblastoma), larynx, stomach,
colon and breast. Therefore, in various embodiments, the NK-1
receptor-binding agent delivery conjugates may be used to treat
and/or diagnose disease states and/or tissues characterized by the
presence of a pathogenic cell population having accessible NK-1
receptors that are uniquely expressed, overexpressed, or
preferentially expressed by the pathogenic cells (i.e., not present
or present at lower concentrations on non-pathogenic cells).
Selective elimination, tracing, or imaging of pathogenic cells may
therefore be mediated by the binding of the NK-1 receptor-binding
moiety to the NK-1 receptor.
[0082] Specifically, over-expression of NK-1 receptors has been
reported in lethal cancers of the brain (glioblastoma, glioma and
astrocytoma), skin (melanoma), pancreas, retina (retinoblastoma),
nerve (neuroblastoma), larynx, stomach, colon and breast, the
various embodiments may be used to develop an NK1R-targeted NIR dye
for use in cancer. Specifically, because NK-1 receptor-binding
agent delivery conjugates are typically unable to cross the blood
brain barrier, they may fail to reach the vast majority of NK-1
receptors present in normal brain tissues. Instead, an NK-1
receptor-binding agent delivery conjugate may selectively
accumulate in NK1R-expressing tumor xenografts with very high
affinity, allowing targeted delivery of the active agents to NK-1
receptor expressing tumor cells.
[0083] In the various embodiments, the NK-1 receptor-binding moiety
may be any of a number of ligands, such as a small molecule ligand
customized to be highly selective for NK-1 receptors. For example,
the small molecule ligand may have a dissociation constant (KD) of
around 13 nM for the NK-1 receptor.
[0084] An example NK-1 receptor-binding ligand ("NK1RL") may be
synthesized from a high affinity NK-1 receptor antagonist
(2S,3S)-3-{[3,5-bis(trifluoromethyl)benzyl]oxy}-2-phenylpiperidine
("L733060" and acetic acid (AcOH), and has the formula:
##STR00006##
[0085] In a first set of embodiments, the active agent of the NK-1
receptor-binding agent delivery conjugate may be a drug for therapy
in order to treat NK-1 receptor positive tumor bearing tissue, as
may be shown in mouse xenograft models expressing NK-1 receptors.
Example drugs may include, but are not limited to, tubulysin B
hydrazide (TubH)) and disulfide-activated desacetyl vinblastine
hydrazide (DAVBH). In such drug conjugates, an example linker group
may include PEG2 coupled to a peptide that contains a chelating
portion of etarfolatide. In particular, the linker group may be
PEG2-Arg-Asp-Lys-2,3 diaminopropionic acid (DAP)-Asp-Cys (referred
to herein as "EC20 peptide" linker), which has a formula of:
##STR00007##
[0086] In another example drug conjugate, the linker group may be
synthesized from a cysteine-based resin, similar to the EC20
peptide linker, but may contain PEG2 that is coupled to a shorter
peptide compared to the EC20 peptide linker. In particular, the
linker group may be PEG2-Arg-Asp-Cys (referred to herein as "Cys
peptide" linker), which has a formula of:
##STR00008##
[0087] In another example drug conjugate, the linker group may be
synthesized from a cysteine-based resin, similar to the EC20
peptide linker and Cys peptide linker, but may contain any of a
variety of PEG structures coupled to a shorter peptide that
contains the chelating portion of etarfolide. In particular, the
linker group may be PEG,-2,3 diaminopropionic acid (DAP)-Asp-Cys
(referred to herein as "short EC20" linker). For example, a short
EC20 linker may include PEG2 (PEG2-based short EC20 linker) and
have a formula of:
##STR00009##
[0088] Another example PEG short EC20 linker may include PEG 12
(PEG12-based short EC20 linker) and have a formula of:
##STR00010##
[0089] Another example PEG short EC20 linker may include PEG36
(PEG36-based short EC20 linker) and have a formula of:
##STR00011##
[0090] In another example, the linker group may contain one or more
peptide sugar (PS) chains in addition to the short EC20 linker. For
example, the linker group may include two PS chains coupled to a
PEG2-based short EC20 linker. Such linker group (PS2-PEG2-based
short EC20 linker) may have a formula of:
##STR00012##
[0091] In another example, the linker group may include a short
EC20 linker that is free of PEG (i.e., PEGO-based short EC20
linker), which is coupled to one or more PS chain. Such linker
group (PS2-PEGO-based short EC20 linker) may have a formula of:
##STR00013##
[0092] In another set of embodiments, the active agent of the NK-1
receptor-binding agent delivery conjugate may be a radioimaging
conjugate. For example, the active agent (i.e., radioimaging agent)
may be a radiotagged element for use in single-photon emission
computed tomography (SPECT) and/or positron emission tomography
(PET) imaging of tumor tissue, and may be shown in mouse xenograft
models expressing NK-1 receptors.
[0093] In some embodiment radioimaging conjugates, the active agent
may be a radiopharmaceutical containing a chelator linked to a
radiotag, such as technetium-99m (.sup.99.sup.mTc). ".sup.mTc is a
metastable nuclear isomer of technetium-99 (.sup.99Tc), and is the
most commonly used medical radioisotope. When used as a radioactive
tracer, .sup.99.sup.mTc can be detected in the body by medical
equipment (gamma cameras). .sup.99.sup.mTc emits readily detectable
140 keV gamma rays (these 8.8 pm photons are about the same
wavelength as emitted by conventional X-ray diagnostic equipment)
and its half-life for gamma emission is 6.0058 hours (meaning 93.7%
of it decays to .sup.9.sup.9Tc in 24 hours). The "short" physical
half-life of the .sup.99.sup.mTc isotope and its biological
half-life of 1 day (in terms of human activity and metabolism)
allow for scanning procedures that collect data rapidly but keep
total patient radiation exposure low. The same characteristics make
the isotope suitable only for diagnostic but never therapeutic
use.
[0094] In other radioimaging conjugates, the active agent may
include a radionuclide suitable for use as a tracer in PET imaging.
For example, Copper-64 (.sup.64Cu) is a positron emitter that may
be well suited for in vitro and in vivo characterization of peptide
probes. Radioimaging conjugates in which .sup.64Cu labeling is used
may also be applied to other copper isotopes and transition metal
isotopes for the purposes of radionuclide imaging. In some
embodiments, a radioimaging agent may be a .sup.64Cu radio-labeled
chelator of 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA). An
example radioactive imaging conjugate may include NK1RL as the NK-1
receptor binding moiety, a linker, and a .sup.6.sup.4Cu-labeled
NOTA group as the active agent, according to the formula:
##STR00014##
[0095] Other compounds may be used in the radioimaging agent and
may be labeled with alternative isotopes for PET imaging instead of
.sup.6.sup.4Cu, including but not limited to, 1 l-indium,
18-fluorine, 68-gallium, etc. For example, in another embodiment
the radioimaging agent may be an .sup.111in radio-labeled chelator
of "4,7,10-tetraazacyclododecane-1,4,7,10-triacetic acid (DOTA).
The radioactive imaging conjugate may include NKIRL as the NK-1
receptor binding moiety, a PEG2 linker, and a .sup.1"in-labeled
DOTA group as the active agent, according to the formula:
##STR00015##
[0096] In an example radioimaging conjugate, the NK-1 receptor
binding moiety may be NK1RL, the linker group may be a short EC20
linker (e.g., PEG2-based short EC20 linker), and the active agent
may be a .sup.99.sup.mTc radiotag, linked to the chelating portion
of the linker group, according to the structure:
##STR00016##
[0097] Alternatively, the linker group may be a different short
EC20 linker (e.g., PEG36-based short EC20 linker), and the active
agent may be .sup.9.sup.9mTc linked to the chelating portion of the
linker group, according to the structure:
##STR00017##
[0098] In another example radioimaging conjugate, the NK-1 receptor
binding moiety may be NKIRL, the linker group may be PS2 coupled to
a short EC20 linker (e.g., PEG2-based short EC20 linker), and the
active agent may be a radiotag, such as technetium-99m
(.sup.99mTc), linked to the chelating portion of the linker group,
according to the structure:
##STR00018##
[0099] In another set of embodiments, the active agent of the NK-1
receptor-binding agent delivery conjugate may be a fluorescent
compound (i.e., fluorescent agent) for use in optical imaging and
fluorescence-guided surgery of certain tumors, as may be shown in
live tumor-bearing mice in which NK-1 receptors are expressed in
tumor tissue. Although most fluorescent agent operate in visible or
ultraviolet parts of the spectrum, near infrared (NIR) area may be
better suited for fluorescence detection and imaging for scenarios
in which high signal-to-noise ratio is important. Therefore, a
fluorescent imaging conjugate may include any of a variety of
fluorescent agents.
[0100] In various embodiments, in tumor uptake in malignant
lesions, specificity of the NK-1 receptor-binding agent delivery
conjugates to the NK-1 receptor may be confirmed using an active
agent containing a fluorescent agent. In particular, fluorescent
visualization of the tumor cells may be performed using a
fluorescent imaging conjugate, and NK-1 receptor specificity may be
established by demonstrating blockade of the fluorescence through
administration of excess unlabeled NK-1 receptor binding moiety
(e.g., NKIRL). In vivo and in vitro studies of the various
embodiment conjugates may be performed using HEK 293 cells which
are transduced with TACR1 to express NK-1 receptors.
[0101] In another example fluorescent imaging conjugate, the NK-1
receptor binding moiety may be NK1RL, the linker group may be a PEG
structure (e.g., PEG36), and the fluorescent agent may be an NIR
fluorescent dye S0456, according to the structure:
##STR00019##
[0102] In another example fluorescent imaging conjugate, the NK-1
receptor binding moiety may be NKIRL, the linker group may be the
EC20 peptide linker, and the fluorescent agent may be an NIR
fluorescent dye LS288-maleimide, according to the structure:
##STR00020##
[0103] In alternative embodiment fluorescent imaging conjugates,
the NK-1 receptor-binding moiety may be a small molecule ligand
other than NKIRL. For example, the NK-1 receptor-binding moiety may
be a sulfur pentafluoride-containing small molecule ("NK1RL-SF5")
according to one of the following structures:
##STR00021##
[0104] In another example fluorescent imaging conjugate, the NK-1
receptor-binding moiety may be NK1RL-SF5, and the linker group may
be PEG2 coupled to a tyrosine-containing peptide, the group being
referred to herein as a "tyrosine peptide linker." The active agent
may be the NIR fluorescent dye S0456, providing an NK-1
receptor-binding moiety may according to the structure:
##STR00022##
[0105] In another example fluorescent imaging conjugate, the NK-1
receptor-binding moiety may be NK1RL-SF5, and the linker group may
be a PEG and a peptide group. For example the linker group may be
PEG2 coupled to a lysine-containing peptide, with the group being
referred to herein as a "lys peptide linker." The active agent may
be a rhodamine compound, providing an NK-1 receptor-binding moiety
may according to the structure:
##STR00023##
[0106] In particular, rhodamine dyes may be used, for example, as a
tracer within water to determine the rate and direction of flow and
transport. Further, since rhodamines may be detected easily and
inexpensively with fluorometers, embodiment fluorescent imaging
conjugates in which the active agent in rhodamine may be used in a
variety of biotechnology applications (e.g., fluorescence
microscopy, flow cytometry, fluorescence correlation spectroscopy,
ELISA, etc.) as part of other processes to develop, test, and
quantify effectiveness of various conjugates.
[0107] Non-limiting examples of fluorophores suitable for use as an
active agent in other embodiments may include, without limitation,
N,N-dimethyl-4-benzofurazansulfonamide (DBD),
4-(2-Aminoethylamino)-7-(N,N-dimethylsulfamoyl)benzofurazan
(DBD-ED), indocyanine green (ICG), a Dylight-700 such as
Dylite-700-2B, IR820; 3,3'-Diethylthiatricarbocyanine iodide
(DTTCI), LS277, a cypatem, and a coumarin.
[0108] In an embodiment, the active agent may be a drug that
includes a nitrogen atom, and the linker group may include
haloalkylenecarbonyl, optionally substituted with a substituent
X.sup.2, where the haloalkylenecarbonyl may be bonded to the drug
nitrogen to form an amide.
[0109] In another embodiment, the active agent may be a drug that
includes an oxygen atom, and the linker group may include
haloalkylenecarbonyl, optionally substituted with a substituent X2,
where the haloalkylenecarbonyl may be bonded to the drug oxygen to
form an ester.
[0110] In another embodiment, the active agent may be a drug that
includes a double-bonded nitrogen atom, and the linker group may
include alkylenecarbonylamino and
1-(alkylenecarbonylamino)succinimid-3-yl, where the linker group
may be bonded to the drug nitrogen to form a hydrazone.
[0111] The drug can include a double-bonded nitrogen atom, and in
this embodiment, the releasable linkers can be
alkylenecarbonylamino and 1-(alkylenecarbonylamino)succinimid-3-yl,
and the releasable linker can be bonded to the drug nitrogen to
form an hydrazone.
[0112] The various embodiments may be understood by reference to
the following non-limiting examples, which are provided by way of
illustration only.
Example 1
[0113] General
[0114] NK-1 receptor-binding agent delivery conjugates were
developed for clinical and diagnostic application. In these
processes, moisture and oxygen sensitive reactions were carried out
under an argon atmosphere. Solid phase peptide synthesis (SPPS) was
performed using a standard peptide synthesis apparatus (Chemglass,
Vineland, N.J.). Column chromatography was performed with silica
gel as the solid phase and TLC was conducted on silica gel TLC
plates and visualized under UV light. All peptides and their
conjugates were purified by preparative reverse phase (RP)-high
performance liquid chromatography (HPLC) and were analyzed by
analytical RP-HPLC. .sup.1H and .sup.13C NMR spectra were acquired
with Bruker 400 or 500 MHz NMR spectrophotometer and the signals
are recorded in ppm with reference to residual CHCI3 (7.27 ppm) or
DMSO (2.50 ppm) and data are reported as s=singlet, d=doublet,
t=triplet, q=quartet, m=multiplet, b=broad with coupling constants
in Hz.
[0115] Electrospray ionization-high resolution mass spectrometry
(ESI-HRMS) was performed utilizing the appropriate polypropylene
glycol standards. Radioactivity was counted on a Packard
.gamma.-counter (Packard Instrument Company, Meriden, Conn.). The
tumor imaging was performed using a Kodak Image Station (In-Vivo
FX, Eastman Kodak Company, New Haven, Conn.).
[0116] HEK 293 cell lines stably transfected with neurokinin-I
receptor (NK1R) were utilized. Cell lines were cultured in
Dulbecco's modified Eagle's medium (GIBCO) supplemented with 10%
fetal bovine serum, G4 18 disulfate (Sigma Aldrich 400 .mu.g/mL),
and 1% penicillin streptomycin at 37.degree. C. in a humidified 95%
air 5% C0.sub.2atmosphere.
[0117] Athymic male nu/nu mice were purchased from Harlan
Laboratories (Indianapolis, Ind.), maintained on normal rodent chow
and housed in a sterile environment on a standard 12h light and
dark cycle for the duration of the study. All animal procedures
were approved by the Purdue Animal Care and Use Committee (PACUC)
in accordance with NIH guidelines.
[0118] Synthesis of NK1RL
[0119] The NK-1 receptor-binding moiety was an NK-1 receptor ligand
(NK1RL) formed from the starting compound
(2S,3S)-3-((3,5-bis(trifluoromethyl)benzyl)oxy)-2-phenylpiperidine
(L-733, 060), a high affinity NK-1 receptor antagonist. The
L-733,060 compound was formed according to the literature
procedure. Formation of NK1RL from L-733,060 was performed
according to the following steps, illustrated in FIG. A.
[0120] To the
(2S,3S)-3-((3,5-bis(trifluoromethyl)benzyl)oxy)-2-phenylpiperidine
(1, 0.065 g, 0.16 mmol) in dry THF (1.5 mL), were added tri
ethylamine (0.056 mL, 0.4 mmol, 2.5 equiv) followed by
tert-butyl-2-bromo acetate (0.035 mL, 0.24 mmol, 1.5 equiv) under
N2. The reaction was stirred for 16 hours at room temperature. The
reaction was quenched with water and 2% HCl solution and extracted
with EtOAc (3.times.5 mL). The combined organic layers were washed
with brine, dried (Na.sub.2S0.sub.4), filtered and concentrated and
residue was purified by silica-gel column chromatography
(hexance:EtOAc, 4:1) to give product, 2 (0.075 g, 92%).
[0121] To the ester (0.075 g, 0.14 mmol) in dry CH.sub.2Cl.sub.2
was added trifluoroacetic acid (TFA) (20 equiv) and stirred for 4
hours at room temperature. The excess of TFA was removed and
diluted with water, extracted with CH2Cl2 (3.times.5 mL). The
combined organic layers were washed with brine, dried
(Na.sub.2S0.sub.4) and concentrated. The residue obtained was
purified by flash silica-gel column chromatography (hexane:EtOAc,
3:7) to give acid, 3 (0.06 g, 90/6) as a white solid.
[0122] Synthesis of NK1RL-EC20 Peptide Linker
[0123] An EC20 peptide linker was created from PEG2 and an amino
acid chain (Arg-Asp-Lys-DAP-Asp-Cys). A compound of the linker and
NK1 receptor-binding moiety NK1RL (NK1RL-EC20 peptide linker) was
synthesized through solid phase peptide synthesis (SPPS) and
purified. Synthesis and purification of the NK1RL-EC20 peptide
linker compound was performed according to the following steps.
[0124] H-Cys(4-methoxytrityl)-Wang resin (150 mg, 0.64 mmol) was
swollen in dichloromethane (2.times.5 mL) and dimethylformamide
(DMF) (2.times.3 mL) while bubbling under argon. After swelling the
resin in DMF, a solution of fluorenylmethyloxycarbonyl
chloride-aspartic acid-4-tert-butyl ester (Fmoc-Asp(OtBu)-OH) (2.5
equiv), benzotriazol-1-yl-oxytripyrrolidinophosphonium
hexafluorophosphate (PyBOP) (2.5 equiv), and
N,N-diisopropylethylamine (DIPEA) (5 equiv) in DMF were added. The
resulting solution was bubbled under argon for 4 hours and drained,
and the resin was washed with DMF (3.times.5 mL) and isopropyl
alcohol (i-PrOH) (3.times.5 mL). Fmoc deprotection was carried out
using 20% piperidine in DMF (3.times.10 mL) and the resin was
washed with DMF (3.times.5 mL) and i-PrOH (3.times.5 mL). Ninhydrin
tests as described in Kaiser et al. "Color test for detection of
free terminal amino groups in the solid-phase synthesis of
peptides," Anal Biochem. 1970 April; 34(2):595-8 ("Kaiser tests")
were conducted to assess coupling and deprotection steps. The above
sequence was repeated for 5 more coupling steps. Final coupling was
done by L-733,060-AcOH (3, 1.5 equiv) under same conditions for 12
hours.
[0125] The resin was washed with DMF (3.times.5 mL) and i-PrOH
(3.times.5 mL) and allowed to dry under nitrogen. The peptide
linker was then cleaved from the resin using a mixture of
trifluoroacetic acid (TFA):3/40:triisopropylsilane:ethanedithiol
cocktail (92.5:2.5:2.5:2.5). The solution was bubbled twice under
nitrogen for 15 min, drained, concentrated, and then precipitated
by addition of cold diethyl ether. Crude product was collected by
centrifugation, washed three more times with cold diethyl ether,
dried under vacuum, and then purified by preparative reverse-phase
HPLC (Waters, XBridge.TM. Prep C18, 5 .mu.m; 19.times.100 mm
column, mobile phase A=20 mM ammonium acetate buffer, pH 5,
B=acetonitrile, gradient 10-100% B in 30 min, 13 mL/min,
.lamda.=280 nm). Pure fractions were analyzed by liquid
chromatography mass spectrometry (LC-MS) and low resolution mass
spectrometry (LR-MS) and were pooled and lyophilized to furnish a
compound of NK1RL-EC20 peptide linker (yield=63.80 mg, 50%. LR-MS
(m/z): 1323.53 M+H)+. UV/vis: Xmax=254 nm). The ligand-linker
compound formed from NK1RL and the EC20 peptide linker is shown in
the schematic equation below:
##STR00024##
[0126] Synthesis of NKIRL-Cys Peptide Linker
[0127] A Cys peptide linker was created from PEG2 and an amino acid
chain (Arg-Asp-Cys). The ligand-linker compound of NKIRL-Cys
peptide linker was synthesized through SPPS with fewer amino acids
starting from Cysteine resin and purified. Synthesis and
purification of NKIRL-Cys was performed using substantially the
same procedure as for NK1RL-EC20 peptide linker.
[0128] Synthesis of Drug Conjugates
[0129] NK1RL-EC20peptide Unker-TubH
[0130] To a solution of NK1RL-EC20 peptide linker (2.80 mg, 2.1 15
umol) in dry DMSO (0.1 mL) at 0.degree. C.,
disulfide-activated-TubH (2.0 mg, 2.115 .mu.mol) followed by DIPEA
(3 .mu.L, 0.21 15 .mu.mol) were added. The reaction mixture was
stirred for 3 hours at room temperature, and the crude product was
purified by RP-HPLC (Waters, XBridge.TM. Prep C18, 5 pam;
19.times.100 mm column, mobile phase A=20 mM ammonium acetate
buffer, pH 7, B=acetonitrile, gradient 10-100% B in 30 min, 13
mL/min,)=280 nm). Pure fractions were analyzed by LC-MS and LR-MS
and were pooled and lyophilized to afford NK1RL-EC20 peptide
linker-TubH. Yield=3.26 mg, 68%. LR-MS (m/z): 2270.53 (M+H)+.
UV/vis: Xmax=254 nm.
[0131] A schematic equation showing the formation of NK1RL-EC20
peptide linker-TubH is shown below:
##STR00025##
[0132] NKIRL-Cys Peptide Linker-TubH
[0133] Further, the same procedural steps were followed using
NKIRL-Cys peptide linker discussed above in order to synthesize an
NKIRL-Cys peptide linker-TubH compound which was then and purified
through RP-HPLC accordingly as described above and
characterized.
[0134] A schematic equation showing the formation of NKIRL-Cys
peptide linker-TubH is shown below:
##STR00026##
[0135] NK1RL-EC20peptide linker-DAVBH
[0136] To a solution of NK1RL-EC20 linker (1.30 mg, 0.9819 .mu.mol)
in dry DMSO (0.1 mL) at 0.degree. C., disulfide-activated-DAVBH
(1.16 mg, 1.1782 .mu.mol) followed by DIPEA (2.5 uL, 19.637 umol)
were added. The reaction mixture was stirred for 4 hours at room
temperature, and the crude product was purified by RP-HPLC (Waters,
XBridge.TM. Prep C18, 5 .mu.m; 19.times.100 mm column, mobile phase
A=20 mM ammonium acetate buffer, pH 7, B=acetonitrile, gradient
10-100% B in 30 min, 13 mL/min, .lamda.=280 run). Pure fractions
were analyzed by LC-MS and LR-MS and were pooled and lyophilized to
afford NK1RL-EC20 peptide linker-DAVBH (yield=1.40 mg, 65%. LR-MS
(m/z): 2195.43 (M+H)+. UV/vis: .lamda.max=254 nm).
[0137] A schematic equation showing the formation of NK1RL-EC20
peptide linker-DAVBH is shown below:
##STR00027##
[0138] NKIRL-Cys Peptide Linker-DAVBH
[0139] Further, the same procedural steps were followed using
NKIRL-Cys peptide linker discussed above in order to synthesize an
NKIRL-Cys peptide linker-DAVBH conjugate which was then and
purified through RP-HPLC accordingly as described above and
characterized.
[0140] A schematic equation showing the formation of NKIRL-Cys
peptide linker-DAVBH is shown below:
##STR00028##
[0141] Cytotoxicity Study (1050) for Drug Conjugates
[0142] HEK 293 NK1R cells (50,000 cells/well) were seeded into 24
well plates (BD Purecoat Amine, BD Biosciences) and allowed to grow
to form monolayer over 24 to 48 hours. The old medium was replaced
with fresh medium (0.5 mL) containing increasing concentrations of
drug conjugates (either targeted or non-targeted NK1RL, and
free-ligand) and cells were incubated for an additional 2 hours at
37.degree. C. Cells were washed (3.times.0.5 mL) with fresh medium
and incubated in fresh medium (0.5 mL) for another 66 hours at
37.degree. C. The spent medium in each well was replaced with fresh
medium (0.5 mL) containing [3H]-thymidine (1 mCi/mL), and the cells
were incubated for additional 4 hours at 37.degree. C. to allow
[3H]-thymidine incorporation. The cells were then washed with
medium (2.times.0.5 mL) and treated with 5% trichloroacetic acid
(0.5 mL) for 10 min at room temperature. The trichloroacetic acid
was replaced with 0.25 N NaOH (0.5 mL), cells were transferred to
individual scintillation vials containing Ecolume scintillation
cocktail (3.0 mL), mixed well to form homogeneous liquid and
counted in a liquid scintillation analyzer. ICso values were
calculated by plotting % [3H]-thymidine incorporation versus log
concentration of drugs (targeted and non-targeted) using in
GraphPad Prism 4.
[0143] In vivo Studies for Drug Conjugates
[0144] Four- to six-week old male nu/nu mice were maintained on a
standard 12 hours light-dark cycle and fed on normal mouse chow for
the duration of the experiment and were inoculated subcutaneously
on their shoulders with HEK 293-NK1R cells (5.0.times.106
cells/mouse in 50% HC Matrigel) using a 25-gauge needle. Growth of
the tumors was measured in two perpendicular directions every 2
days using a caliper, and the volumes of the tumors were calculated
as 0.5.times.L.times.W2 (L=measurement of longest axis, and W=axis
perpendicular to L in millimeters). Experiments on live mice
involved at least five mice per group and animals were treated with
therapeutic drug conjugates (1.6 .mu.mol/kg of body weight) in
saline (100 .mu.L) for three weeks, 3 doses per week (M/W/F), when
the tumors reached 75-130 mm.sup.3 volume (.about.3 weeks). Tumor
volumes and body weights were also measured at each dose. In vivo
efficacy was evaluated by plotting tumor volume versus days and %
of weight loss/gain versus days on therapy. Three weeks after
treatment, the animals were dissected and selected tissues were
preserved in formalin for histopathology studies. H&E stained
slides for microscopic evaluation were prepared from submitted
fixed tissues.
[0145] FIG. 1B provides in vivo mice therapeutic data for HEK
293-NK1R tumor xenograft models showing behavior of tumor volumes
from the NK1R-EC20 peptide linker-TubH conjugate. FIG. 1C provides
in vivo mice therapeutic data on HEK 293-NK1R tumor xenografts
model showing behavior body weights during the therapy shown in
FIG. 1B.
Example 2
[0146] As illustrated in FIG. 2, synthesis of NKIRL-Lys peptide
linker-rhodamine conjugate was carried out by synthesizing the
NKIRL-Lys peptide linker, to which NHS-rhodamine was added.
Specifically, NKIRL-Lys peptide linker-rhodamine conjugate
synthesis was performed using the following steps.
[0147] Synthesis of NKIRL-Lys Peptide Linker
[0148] H-Lys (Boc)-2-Cl-Trt resin (80 mg, 0.75 mmol) was swollen in
dichloromethane (DCM) (2.times.5 mL) and DMF (2.times.3 mL) while
bubbling under argon. A solution of Fmoc-Asp(OtBu)-OH (2.5 equiv),
PyBOP (2.5 equiv), and DIPEA (5 equiv) in DMF was added. The
resulting solution was bubbled under argon for 3 hours and drained,
and the resin was washed with DMF (3.times.5 mL) and i-PrOH
(3.times.5 mL). Fmoc deprotection was carried out using 20%
piperidine in DMF (3.times.5 mL). The deprotection solution was
removed, and the resin was washed again with DMF (3.times.5 mL) and
i-PrOH (3.times.5 mL). Kaiser tests were conducted to assess
coupling and deprotection steps. The same procedure were followed
for
Fmoc-arginine-(2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-sulfonyl)
ester (Fmoc-Arg(pbf)-OH), Fmoc-PEG2-CO 2H, and NKIL
(L-733,060-acetic acid) couplings, with the reaction being bubbled
over night for NK1L. The resin was washed with DMF (3.times.5 mL)
and i-PrOH (3.times.5 mL) and allowed to dry under nitrogen. The
NKIL-peptide linker was then cleaved from the resin using a mixture
of 95% trifluoroacetic acid (TFA), 2.5% H.sub.20 and 2.5%
triisopropylsilane (TIPS). The solution was bubbled three times
under nitrogen for 15 min, drained, concentrated, and then
precipitated by addition of cold diethyl ether. Crude product was
collected by centrifogation, washed three more times with cold
diethyl ether, dried under vacuum, and then purified by preparative
reverse-phase HPLC (Waters, XBridge.TM. Prep C18, 5 prn;
19.times.100 mm column, mobile phase A=20 mM ammonium acetate
buffer, pH 7, B=acetonitrile, gradient 0-80% B in 30 min, 13
mL/min, .lamda.=254 nm). Pure fractions were analyzed by LC-MS
(XBridge.TM. RP18, 3.5 .mu.m; 3.0.times.50 mm column) and low
resolution electrospray ionisation mass spectrometry (LR-ESIMS),
and were pooled and lyophilized to finish NKIRL-Lys peptide linker
(Compound 1 in FIG. 2).
[0149] Synthesis of NK1RL-Lys Peptide Linker-Rhodamine
Conjugate
[0150] The purified NK1RL-Lys peptide linker (Compound 1 in FIG. 2)
was coupled with NHS-rhodamine by stirring 1:1.2 ratios of Compound
I to NHS-rhodamine in dry DMSO. DIPEA under argon for 12 hours at
room temperature. The resulting material was purified using RP-HPLC
(mobile phase A=20 mM ammonium acetate buffer, pH 7,
B=acetonitrile, gradient 0-80% B in 30 min, 13 mL/min, .lamda.=280
nm). Pure fractions were combined, concentrated under vacuum, and
lyophilized to yield the product, NK1RL-Lys peptide
linker-rhodamine (Compound 2 in FIG. 2). The NK1RL-Lys peptide
linker-rhodamine conjugate is a reddish solid, was analyzed by
LC-MS and LR-ESIMS.
[0151] Synthesis of NK1RL-EC20 Peptide Linker-LS288 Conjugate
[0152] NK1RL was synthesized as the NK-1 receptor-binding moiety as
discussed above. Synthesis of NK1RL-EC20 peptide linker-LS288
conjugate is illustrated in FIG. 3, and was carried out using the
following steps.
[0153] To the NK1RL-EC20 peptide linker (Linker 7) in dry DMSO were
added LS288-maleimide (Compound 8) followed by DIPEA under argon
atmosphere at room temperature. The reaction mixture was stirred
for overnight at room temperature. The product was precipitated by
addition of isopropanol and collected by centrifugation. The crude
product was purified by preparative reverse phase HPLC using a
mobile phase of A=20 mM ammonium acetate buffer, pH 7;
B=acetonitrile; gradient 0-50% B in 30 min, 13 mL/min, .lamda.=280
nm. Pure fractions were analyzed by LC-MS and LR-MS and were pooled
and lyophilized to furnish NK1RL-EC20 peptide linker-LS288
conjugate (Conjugate 9).
[0154] Fluorescent Confocal Microscopy Imaging
[0155] HEK 293-NK1R cells (50,000 cells/well in 0.5 mL) were seeded
into confocal microwell plate (Lab-Tek, Chambered #1.0 Borosilicate
Coverglass) and allowed cells to form monolayers over 24 hours.
Spent medium was replaced with fresh medium containing NK1RL-Lys
peptide linker-rhodamine (25 nM) in the presence or absence of
100-fold excess free ligand and cells were incubated for 1 hour at
37.degree. C. After washing with fresh medium (3.times.0.5 mL),
confocal images were acquired using a confocal microscopy (FV 1000,
Olympus).
[0156] FIG. 4 shows resulting images from the binding studies of
the NK1RL-Lys peptide linker-rhodamine conjugate to HEK 293-NK1R
cells. Specifically, the images show incubation of cells for 1 hour
at 37.degree. C. in the absence (a) and presence (d) of 100-fold
excess of competing agent (NK1RL alone) to the conjugate at 25 nM
concentration. The images in (b) are 3.times. magnification from
(a), and in (c) are 3.times. magnification of white light.
[0157] Flow Cytometric Analysis
[0158] Procedure
[0159] HEK 293 NK1R cells were seeded into a T75 flask and allowed
to form a monolayer over 48 hours. After trypsin digestion,
released cells were transferred into centrifuge tubes (1.times.105
cells/tube) and centrifuged. The medium was replaced with fresh
medium containing NKIRL-Rhod (25 nM) in the presence or absence of
100-fold excess unlabeled NK1RL ligand and incubated for 1h at
37.degree. C. After rinsing with fresh medium (3.times.0.5 mL),
cells were re-suspended in PBS (0.5 mL) and cell bound fluorescence
was analyzed (40,000 cells/sample) using a flow cytometer.
Untreated HEK 293-NK1R cells in PBS served as a negative
control.
[0160] Determination if Binding Affinity and Specificity
[0161] HEK 293 NK1R cells (50,000 cells/well) were seeded into 24
well plates (BD Purecoat Amine, BD Biosciences) and allowed to grow
to confluence over 48-72 hours. Spent medium in each well was
replaced with 0.5 mL of fresh medium containing 0.5% bovine serum
albumin and increasing concentrations of the NIR dye conjugates in
the presence or absence of 100-fold excess of competing ligand
(i.e., L-733,060). After incubation for 1 hour at 37.degree. C.
cells were rinsed with incubation solution (2.times.0.5 mL) to
remove unbound fluorescence and dissolved in 0.5 mL of 1% aqueous
sodium dodecyl sulfate (SDS). Cell associated fluorescence was then
determined by measuring maximum emission of the resulting solution
by transfer to a quartz cuvette upon excitation of each dye
(rhodamine/LS288) at 545/755 ran using an Agilent Technologies Cary
Eclipse fluorescence spectrophotometer. Experiments were performed
in triplicate. The conjugate's dissociation constant (KD) was
calculated from a lot of cell bound fluorescence emission (a.u.)
versus the concentration of targeted NIR probe added using the
GraphPad Prism 4 program and assuming a non-cooperative single site
binding equilibrium.
[0162] FIG. 5 shows the binding of NKIRL-Lys peptide
linker-rhodamine to HEK 293-NK1R cells by flow cytometry. FIG. 6A
shows the binding affinity of the NK1RL-Lys peptide
linker-rhodamine conjugate in cultured HEK 293-NK1R cells
expressing NK-1 receptor. FIG. 6B shows the binding affinity of the
NK1RL-EC20 peptide linker-LS288-maleimide conjugate.
[0163] In Vivo Assays
[0164] Implantation if Subcutaneous Tumors Using HEK293-NK1R
Cells
[0165] Six week old male athymic nu/nu mice (Harlan Laboratories,
IN) were inoculated subcutaneously on their shoulders with HEK 293
NK1R cells (5.0.times.106 cells/mouse in 50% HC Matrigel) using a
25-gauge needle. Growth of the tumors was measured in two
perpendicular directions every 2 days using a caliper, and the
volumes of the tumors were calculated as 0.5.times.L*W2
(L=measurement of longest axis, and W=measurement of axis
perpendicular to L in millimeters). Animals were imaged when the
tumors reached 300-500 mm.sup.3 volume (.about.2-3 weeks).
Experiments on live mice involved at least four mice per group.
Imaging was then performed as described below.
[0166] Fluorescence Imaging and Analysis of Mice
[0167] Tumor bearing mice were treated via tail vein (i.v)
injection with 10 nmol of dye conjugate with 100 fold excess
competition [three groups (one is dye, two is competition and three
is -ve control (KB tumor) groups), 4 mice/group] and imaged 2 hours
post injection using a Caliper IVIS Lumina II Imaging station
coupled to ISOON5 160 Andor Nikon camera equipped with Living Image
Software Version 4.0. The 2 hour time point for imaging was chosen
based on data from previous experiments showing that a radio
labeled conjugate of NK1 yielded the highest tumor-to-background
ratio at this time point. The settings were as follows: lamp level,
high; excitation, 745 nM: emission, ICG; epi illumination, binning
(M) 4; FOV, 12.5: f-stop, 4; acquisition time, 1 second. After
completion of whole body imaging, animals were dissected and
selected organs were collected and imaged again for complete
biodistribution. All organs were preserved in 25 mL of formalin in
preparation for submission to the Purdue Histology &
Phenotyping Laboratory for hematoxylin and eosin staining.
[0168] FIG. 6C shows (a-b) HEK 293-NK1R tumor xenograft model mice
treated with conjugates in which LS288 is the active agent, (c-d)
blocking images for the HEK 293-NK1R tumor xenograft model mice
with the treatment in a-b, and (e-f) NK1R-negative tumor xenograft
model mice treated with conjugates in which LS288 is the active
agent. FIG. 6D is a biodistribution study of the imaged mice in
FIG. 6C.
Example 3
[0169] Synthesis of the NK1RL-EC20 Peptide Linker-".sup.mFc
Conjugate
[0170] A solution of sodium pertechnetate (1.0 mL, 15 mCi) was
added to a vial containing a lyophilized mixture of NKIRL-EC20
peptide linker (0.178 mg), sodium a-D-glucoheptanoate dehydrate (80
mg), stannous chloride dihydrate (0.8 mg), and sufficient NaOH to
achieve pH of 7.2 upon rehydration with water. After adding sodium
pertechnetate (15 mCi), the vial was heated in a boiling water bath
for 18 minutes and then cooled to room temperature before use. The
labeling efficiency, radiochemical purity, and radiochemical
stability were analyzed by RP-HPLC.
[0171] In Vitro Studies
[0172] HEK 293 NK1R cells (50,000 cells/well) were seeded into 24
well plates (BD Purecoat Amine, BD Biosciences) and allowed to grow
to confluence over 48-72 hours. Spent medium in each well was
replaced with 0.5 mL of fresh medium containing 0.5% bovine serum
albumin and increasing concentrations of NKIRL-EC20 peptide
linker-.sup.99.sup.mTc in the presence or absence of 100-fold
excess of competing ligand, i.e., L-733,060. After incubation for 1
hour at 37.degree. C. cells were rinsed with incubation solution
(3.times.0.5 mL) to remove unbound radioactive material and cells
were dissolved in 0.25 M NaOH aqueous (0.5 mL) solution. The
dissolved cells were transferred into individual .gamma.-counter
tubes and radioactivity was counted using a .gamma.-counter. The
binding constant (Kd) was calculated by plotting bound
radioactivity versus the concentration of targeted radiotracer
using GraphPad Prism 4 program, illustrated in FIG. 7A.
[0173] In Vivo Studies
[0174] Four- to six-week old male nu/nu mice were inoculated
subcutaneously on their shoulders with HEK 293 NK1R cells
(5.0.times.106 cells/mouse in 50% HC Matrigel) using a 25-gauge
needle. Growth of the tumors was measured in two perpendicular
directions every 2 days using a caliper, and the volumes of the
tumors were calculated as 0.5.times.L*W2 (L=measurement of longest
axis, and W=axis perpendicular to L in millimeters). Animals were
treated with NK1RL-EC20 peptide linker-.sup.99.sup.mTc (1.34 nmol,
150 .mu.Ci) in saline (100 .mu.L) when the tumors reached 300-500
mm3 volume (.about.3 weeks). Experiments on live mice involved at
least four mice per group, animals were sacrificed by C0.sub.2
asphyxiation at different time points as described below. Images
were acquired by a Kodak Imaging Station in combination with CCD
camera and Kodak molecular imaging software (version 4.0)
(radioimages: illumination source=radio isotope, acquisition time=2
and 4 min, f-stop=0, focal plane=5, FOV=162.5, binning=4; White
light images: illumination source=white light transillumination,
acquisition time=0.175 seconds, f-stop=11, focal plane=5, FOV=162.5
with no binning).
[0175] Following imaging, animals were dissected and selected
tissues were collected into pre-weighed .gamma.-counter tubes.
Radioactivity of pre-weighed tissues and NK1RL-EC20 peptide
linker-.sup.99.sup.mTc (1.34 nmol, 150 .mu.Ci) in saline (100 .mu.)
was counted in a .gamma.-counter. CPM values were decay corrected
and results were calculated as % ID/gram of wet tissue and
tumor-to-tissue ratios.
[0176] FIG. 7B is a set of whole body mice images of mice for
NK1RL-EC20 peptide linker-99mTc conjugate, showing on the left a
HEK 293-NK1R tumor xenograft model treated with NK1RL-EC20 peptide
linker-.sup.99.sup.mTc conjugate, and on the right the HEK 293-NK1R
tumor xenograft model aged mouse with blocking.
[0177] FIG. 7C shows results from a biodistribution study of mice
images for the NK1RL-EC20 peptide linker-.sup.99.sup.mTc conjugate.
For each area, the left-most bar corresponds to administration of
NK1RL-EC20 peptide linker-.sup.99.sup.mTc conjugate, and the
right-most bar shows a competitive NK-1 receptor ligand labeled
with ''.sup.mTc. FIG. 7D is a set of whole body mice images on
SPECT-CT for NK1 RL-EC20 peptide linker-99mTc conjugate in HEK
293-NK1R tumor xenograft model mice. FIG. 7E is a set of whole body
mice images on SPECT-CT for NK1RL-EC20 peptide linker-.sup.99mTc
conjugate in NK1R-negative tumor xenograft model mice.
Example 4
[0178] Synthesis of the NK1RL-PEG2-NOTA-Conjugate
[0179] NOTA-NHS ester (11 mg, 0.0016 mmol), followed by DIPEA amine
in dry DMSO under argon were added to a purified NK1RL-PEG2 linker
(10 mg, 0.0016 mmol), created according to procedures described
above. The reaction mixture was stirred for 12 hours at room
temperature. The reaction progress was confirmed by LC-MS and
purified by RP-HPLC (mobile phase A=20 raM ammonium acetate buffer,
pH 7, B=acetonitrile, gradient 10-100% B in 30 minutes, 13 mL/min,
.lamda.=254 nm). Pure fractions were combined, concentrated under
vacuum, and lyophilized to yield the NK1 RL-PEG2-NOTA conjugate,
which was analyzed by LC-MS and LR-ESIMS.
[0180] In Vivo Studies
[0181] A NK-1 receptor-binding radionuclide delivery conjugate was
prepared that contained NK1RL, a PEG2 linker, and {circumflex over
( )}Cu-labeled chelator (radiochemical purity: 99%) with Specific
Activity (SA) of 3.7 MBq{circumflex over ( )}g. Four
NK1R-transduced xenografts (HEK 293-NK1R) were prepared, as well as
four non-transduced xenografts (HEK 293-WT) using athymic nude
mice. Doses of 140-160 uCi (.about.2 nmoles) of ligand per mouse
were intravenously administered, and the models were imaged at 1, 4
and 20 hours post-injection using PET. The images were analyzed in
a region of interest (ROI) around the tumor xenograft activity, and
percentage injected dose per mL (% ID/mL) values were calculated
from the mean activity in the ROIs. FIG. 8A is a plot showing ROI
activity for the NK1R-transduced and non-transduced xenografts.
Therefore, the data showed that the NK1RL-PEG2-NOTA-.sup.6.sup.4Cu
conjugate specifically accumulated in NK1R-transduced xenografts,
but not in non-transduced murine model xenografts. FIG. 8B is a
plot showing the {circumflex over ( )}Cu-NK1R ligand uptake ratio
between the NK1R-transduced and non-transduced xenografts in
various areas at 20 hours post-injection.
[0182] Analysis of these data showed that that the ratio between
NK1R-transduced and non-transduced xenografts was highest at 20
hours post-injection, which may indicate that this radioactive
imaging conjugate is more suitable for imaging at a relatively late
time point after administration.
[0183] FIG. 8C is a set of HEK 293-NK1R tumor xenograft model mice
on PET using a conjugate in which the active agent has
.sup.64Cu.
Example 5
[0184] Synthesis of the NK1RL-PEG36-Based Short EC20
Linker-{circumflex over ( )}Tc Conjugate
[0185] A solution of sodium pertechnetate (1.0 mL, 15 mCi) was
added to a vial containing a lyophilized mixture of NK1RL-EC20
peptide linker (0.178 mg), sodium a-D-glucoheptanoate dehydrate (80
mg), stannous chloride dihydrate (0.8 mg), and sufficient NaOH to
achieve pH of 7.2 upon rehydration with water. After adding sodium
pertechnetate (15 mCi), the vial was heated in a boiling water bath
for 18 minutes and then cooled to room temperature before use. The
labeling efficiency, radiochemical purity, and radiochemical
stability were analyzed by RP-HPLC.
[0186] In Vitro Studies
[0187] HEK 293 NK1R cells (50,000 cells/well) were seeded into 24
well plates (BD Purecoat Amine, BD Biosciences) and allowed to grow
to confluence over 48-72 hours. Spent medium in each well was
replaced with 0.5 mL of fresh medium containing 0.5% bovine serum
albumin and increasing concentrations of NKIRL-PEG36-containing
short EC20 linker-.sup.99mTc in the presence or absence of 100-fold
excess of competing ligand, i.e., L-733,060. After incubation for 1
hour at 37.degree. C., cells were rinsed with incubation solution
(3.times.0.5 mL) to remove unbound radioactive material and cells
were dissolved in 0.25 M NaOH aqueous (0.5 mL) solution. The
dissolved cells were transferred into individual .gamma.-counter
tubes and radioactivity was counted using a .gamma.-counter. The
binding constant (Kd) was calculated by plotting bound
radioactivity versus the concentration of targeted radiotracer
using GraphPad Prism 4 program. FIG. 9A illustrates this binding
data for NK1RL-PEG36-containing short EC20 linker-.sup.99.sup.mTc
in the presence of the competing ligand (top) and without the
competing ligand (bottom).
[0188] In Vivo Studies
[0189] Four- to six-week old male nu/nu mice were inoculated
subcutaneously on their shoulders with HEK 293 NK1R cells (5.0*106
cells/mouse in 50% HC Matrigel) using a 25-gauge needle. Growth of
the tumors was measured in two perpendicular directions every 2
days using a caliper, and the volumes of the tumors were calculated
as 0.5.times.L*W2 (L=measurement of longest axis, and W=axis
perpendicular to L in millimeters). Animals were treated with
NK1RL-PEG36-containing short EC20 linker-.sup.99.sup.mTc (2 nmol,
150 .mu.Ci) in saline (100 .mu.L) when the tumors reached 300-500
mm.sup.3 volume (.about.3 weeks). Experiments on live mice involved
three mice per group, animals were sacrificed by C0.sub.2
asphyxiation at different time points as described below. Images
were acquired by a Kodak Imaging Station in combination with CCD
camera and Kodak molecular imaging software (version 4.0)
(radioimages: illumination source=radio isotope, acquisition time=2
and 4 min, f-stop=0, focal plane=5, FOV=162.5, binning=4; White
light images: illumination source=white light transillumination,
acquisition time=0.175 sec, f-stop=11, focal plane=5, FOV=162.5
with no binning).
[0190] Following imaging, animals were dissected and selected
tissues were collected into pre-weighed .gamma.-counter tubes.
Radioactivity of pre-weighed tissues and NK1RL-PEG36-containing
short EC20 linker-.sup.99.sup.mTc (2 nmol, 150 .mu.Ci) in saline
(100 .mu.L) was counted in a .gamma.-counter. CPM values were decay
corrected and results were calculated as % ID/gram of wet tissue
and tumor-to-tissue ratios.
[0191] FIG. 9B is a set of whole body mice images of mice for
NK1RL-PEG36-containing short EC20 linker-.sup.99.sup.mTc conjugate,
showing in the upper row a HEK 293-NK1R tumor xenograft model group
with a lead shield positioned to allow observation of the relative
radioactivity in only the tumor areas. The lower row shows a HEK
293-NK1R tumor xenograft model group treated with
NK1RL-PEG36-containing short EC20 linker-.sup.99mTc conjugate
without shielding.
[0192] FIGS. 9C and 9D show results from a biodistribution study of
mice images for the NKIRL-PEG36-containing short EC20
linker-''.sup.mTc at 2 hours and 8 hours post-injection,
respectively. For each area, the left-most bar corresponds to
administration of NK1RL-PEG36-containing short EC20
linker-''.sup.mTc conjugate, and the right-most bar shows a
competitive NK-1 receptor-binding ligand (L-733,060) labeled with
.sup.99mTc.
Example 6
[0193] As illustrated in FIG. 10A, synthesis of NKIRL-SF5-Lys
peptide linker-rhodamine conjugate was carried out by synthesizing
the NK1RL-SF5 NK-1 receptor-binding moiety, which was used to
create the NK1RL-SF5-Lys peptide linker, to which NHS-rhodamine was
added. Specifically, NK1RL-SF5-Lys peptide linker-rhodamine
conjugate synthesis was performed using the following steps.
[0194] Synthesis of NK1RL-SF5
[0195] Synthesis of an alternative NK-1 receptor-binding moiety
NK1RL-SF5, discussed above, was carried out using the following
steps.
[0196] Starting with
(2S,3S)-3-(3-pentatrifluoromethyl)benzyloxy)-2-phenylpiperidine (1,
0.055 g, 0.00013 mmol) in dry THF (1.0 mL), were added tri
ethylamine (TEA) (0.048 mL, 0.00034 mmol, 2.5 equiv), followed by
tert-butyl-2-bromo acetate (0.03 mL, 0.00021 mmol, 1.5 equiv) under
N.sub.2. The reaction was stirred for 16 hours at room temperature,
The reaction was quenched with water and 2% HCl solution, and
extracted with ethyl acetate (EtOAc) (3.times.5 mL). The combined
organic layers were washed with brine, dried with sodium sulfate
(Na.sub.2S0.sub.4), filtered, and concentrated. The resulting
residue was purified using silica-gel column chromatography
(hexance:EtOAc, 4:1) to give an intermediary ester (0.070 g, 93%)
according to the formula:
##STR00029##
[0197] TFA (20 equiv) was added to the intermediary ester (0.07 g,
0.00013 mmol) in dry CH2C2, and stirred for 4 hours at room
temperature. The excess of TFA was removed and diluted with water,
extracted with CH2C12 (3.times.5 mL). The combined organic layers
were washed with brine, dried (Na.sub.2SO.sub.4) and concentrated.
The residue obtained was purified by flash silica-gel column
chromatography (hexane:EtOAc, 3:7) to give a product NK1RL-SF5
(0.052 g, 90%).
[0198] Synthesis of NK1RL-SFS-Lys Peptide Linker
[0199] An NK1RL-SF5-Lys peptide linker was synthesized by following
an analogous procedure to that used to synthesize the NK1RL-Lys
peptide linker as discussed above with respect to FIG. 2. The
NK1RL-SF5-Lys peptide linker was then purified through RP-HPLC and
characterized accordingly as described above.
[0200] Synthesis of NK1RL-SF5-Lys Peptide Linker-Rhodamine
Conjugate
[0201] The purified NK1RL-SF5-Lys peptide linker (Compound 1 in
FIG. 10A) was coupled with NHS-rhodamine by stirring 1:1.2 ratios
of the NK1 RL-SF5-Lys peptide linker and NHS-rhodamine in dry DMSO,
DIPEA under argon for 12 hours at room temperature. The resulting
material was purified by RP-HPLC (mobile phase A=20 raM ammonium
acetate buffer, pH 7, B=acetonitrile, gradient 10-100% B in 30 min,
13 mL/min, .lamda.=280 nm). Pure fractions were combined,
concentrated under vacuum, and lyophilized to yield the product,
NK1RL-SF5-Lys peptide linker-rhodamine conjugate (Compound 2 in
FIG. 10A). The NK1RL-SF5-Lys peptide linker-rhodamine conjugate, a
reddish solid, was analyzed using LC-MS and LR-ESIMS.
[0202] FIG. 10B shows the binding affinity of the NK1RL-SF5-Lys
peptide linker-rhodamine conjugate in cultured HEK 293-NK1R cells
expressing NK-1 receptor.
[0203] Fluorescent Confocal Microscopy Imaging
[0204] HEK 293-NK1R cells (50,000 cells/well in 0.5 mL) were seeded
into confocal microwell plate (Lab-Tek, Chambered #1.0 Borosilicate
Coverglass) and allowed cells to form monolayers over 24 hours.
Spent medium was replaced with fresh medium containing
NK1RL-SF5-Lys peptide linker-rhodamine (25 nM) in the presence or
absence of 100-fold excess free ligand and cells were incubated for
1 hour at 37.degree. C. After washing with fresh medium
(3.times.0.5 mL), confocal images were acquired using a confocal
microscopy (FV 1000, Olympus).
[0205] FIG. 10C shows resulting images from the binding studies of
the NK1RL-SF5-Lys peptide linker-rhodamine conjugate to HEK
293-NK1R cells. Specifically, the confocal images provide (a) a
magnification of cells after incubation for 1 hour at 37.degree. C.
in the absence of competing agent, (b) a magnified white light
image of the cells in (a), (c) a magnification of the cells in the
presence of a 100-fold excess of competing agent (NK1RL alone) to
the conjugate at 25 nM concentration, and (d) a magnified white
light image of the cells in (c).
Example 7
[0206] Synthesis of NK1RL-SF5-Tyrosine peptide linker-S0456
[0207] A fluorescent imaging conjugate, NK1RL-SF5-tyrosine peptide
linker-S0456, was synthesized, as illustrated in FIG. 11, according
to the following steps.
[0208] Starting with a mixture of tert-butyl-L-tyrosine (1.1 equiv)
and Fmoc-PEG2-CO .sub.2H in CH2Cl2,
1-[Bis(dimethylamino)methylene]-IH-1,2,3-triazolo[4,5-b]pyridinium
3-oxid hexafluorophosphate (HATU) (1.2 equiv) was added, followed
by DIPEA (5.0 equiv) under nitrogen. The reaction mixture was
stirred for 12 hours at room temperature. The reaction was then
quenched with water and 2% HCl solution, and extracted with EtOAc
(3.times.5 mL). The combined organic layers were washed with brine,
dried using sodium sulfate, filtered, and concentrated. The
resulting residue was purified by silica-gel column chromatography
(hexane-EtOAc, 4:1) to afford a first intermediary (Compound 1 in
FIG. 11), which was confirmed by LC-MS.
[0209] Piperidine in THF (1:1) was added to the pure Compound I
under nitrogen and stirred for 2 hours at room temperature.
Completion of the reaction was confirmed by LC-MS, and an excess of
solvent and piperidine were evaporated, resulting in a second
intermediary (Compound 2 in FIG. 11).
[0210] Compound 2 (1.0 equiv) was mixed with NK1RL-SF5 (1.0 equiv)
in CH.sub.2Cl.sub.2. HATU (1.2 equiv) was added to the mixture,
followed by DIPEA (5.0 equiv) under nitrogen. The reaction mixture
was stirred for 12 hours at room temperature. The reaction was
quenched with water and 2% HCl solution, and extracted with EtOAc
(3.times.5 mL). The combined organic layers were washed with brine,
dried with sodium sulfate, filtered, and concentrated. The residue
was purified by silica-gel column chromatography (hexane:EtOAc,
4:1) to afford a third intermediary (Compound 3 in FIG. 11), which
was confirmed by LC-MS.
[0211] S0456 dye (1.0 equiv) was added to the pure Compound 3 in
DMF, followed by the addition of K2CO3 (5.0 equiv). The reaction
mixture was stirred at 60.degree. C. for 4 hours, and at room
temperature for 12 hours under nitrogen. The progress of the
reaction was monitored and confirmed by LC-MS. After completion of
the reaction, crude mass was filtered to remove all solids, and
filtrate was stirred with TFA for I hour at room temperature. The
tert-butyl ester hydrolysis product was confirmed by LCMS and the
crude mass was purified by RP-HPLC (mobile phase A=20 mM ammonium
acetate buffer, pH 7, B=acetonitrile, gradient 0-80% B in 30 min,
13 mL/min, .lamda.=280 nm). Pure fractions were combined,
concentrated under vacuum, and lyophilized to yield the product,
NK1RL-SF5-tyrosine peptide linker-S0456 (Compound 4 in FIG. 11).
The product Compound 4 was analyzed by LC-MS and LR-ESIMS.
[0212] In Vitro Studies
[0213] HEK 293 NK1R cells (50,000 cells/well) were seeded into 24
well plates (BD Purecoat Amine, BD Biosciences) and allowed to grow
to confluence over 48-72 hours. Spent medium in each well was
replaced with 0.5 mL of fresh medium containing 0.5% bovine serum
albumin and increasing concentrations of NK1RL-SF5-Tyrosine peptide
linker-S0456 in the presence of 100-fold excess of competing
ligand, i.e., L-733,060. The binding constant (Kd) was calculated
by plotting fluorescence versus the concentration of targeted
radiotracer using GraphPad Prism 4 program, illustrated in FIG.
12.
Example 8
[0214] Synthesis of NK1RL-PEG2-DOTA Conjugate
[0215] DOTA-NHS ester (6.8 mg, 0.0135 mmol) was added to a purified
NK1RL-PEG2 linker (8 mg, 0.0135 mmol), followed by the addition of
DIPEA in dry DMSO under argon. The reaction mixture was stirred for
12 hours at room temperature. The reaction progress was confirmed
by LC-MS and purified by RP-HPLC (mobile phase A=20 mM ammonium
acetate buffer, pH 7, B=acetonitrile, gradient 10-100% B in 30 min,
13 mL/min, .lamda.=254 ran). Pure fractions were combined,
concentrated under vacuum, and lyophilized to yield the product,
NK1 RL-PEG2-DOTA, was analyzed by LC-MS and LR-ESIMS.
[0216] In Vivo Studies
[0217] NK1R-transduced xenografts (HEK 293-NK1R) were prepared in
groups, with three mice per group. Doses of around 238 uCi of
ligand per mouse were intravenously administered, and the models
were imaged at 4 hours post-injection using PET, an example of
which is shown in FIG. 13A. The images were analyzed in a region of
interest (ROI) around the tumor xenograft activity, and percentage
injected dose per mL (% ID/mL) values were calculated from the mean
activity in the ROIs. FIG. 13B is a plot showing this ROI activity
for the NK1R-transduced xenografts. FIG. 13C is a whole body mouse
image on SPECT-CT for the NK1RL-PEG2-DOTA-1-11ln conjugate in a HEK
293-NK1R tumor xenograft model mouse.
[0218] The foregoing method descriptions and the process flow
diagrams are provided merely as illustrative examples and are not
intended to require or imply that the steps of the various
embodiments must be performed in the order presented. As will be
appreciated by one of skill in the art the order of steps in the
foregoing embodiments may be performed in any order. Words such as
"thereafter." "then," "next," etc. are not intended to limit the
order of the steps; these words are simply used to guide the reader
through the description of the methods. Further, any reference to
claim elements in the singular, for example, using the articles
"a," "an" or "the" is not to be construed as limiting the element
to the singular.
[0219] Skilled artisans may implement the above-described methods,
processes and/or functionality in varying ways for each particular
application, but such implementation decisions should not be
interpreted as causing a departure from the scope of the present
invention.
[0220] The preceding description of the disclosed embodiments is
provided to enable any person skilled in the art to make or use the
present invention. Various modifications to these embodiments will
be readily apparent to those skilled in the art, and the generic
principles defined herein may be applied to other embodiments
without departing from the spirit or scope of the invention. Thus,
the present invention is not intended to be limited to the
embodiments shown herein but is to be accorded the widest scope
consistent with the following claims and the principles and novel
features disclosed herein.
* * * * *